In vivo structural analysis of articular cartilage using diffusion tensor magnetic resonance imaging

Purpose

The articular cartilage is a small tissue with a matrix structure of three layers between which the orientation of collagen fiber differs. A diffusion-weighted twice-refocused spin-echo echo-planar imaging (SE-EPI) sequence was optimized for the articular cartilage, and the structure of the three layers of human articular cartilage was imaged in vivo from diffusion tensor images.

Materials and Methods

The subjects imaged were five specimens of swine femur head after removal of the flesh around the knee joint, five specimens of swine articular cartilage with flesh present and the knee cartilage of five adult male volunteers. Based on diffusion-weighted images in six directions, the mean diffusivity (MD) and the fractional anisotropy (FA) values were calculated.

Results

Diffusion tensor images of the articular cartilage were obtained by sequence optimization. The MD and FA value of the specimens (each of five examples) under different conditions were estimated. Although the articular cartilage is a small tissue, the matrix structure of each layer in the articular cartilage was obtained by SE-EPI sequence with GRAPPA. The MD and FA values of swine articular cartilage are different between the synovial fluid and saline. In human articular cartilage, the load of the body weight on the knee had an effect on the FA value of the surface layer of the articular cartilage.

Conclusion

This method can be used to create images of the articular cartilage structure, not only in vitro but also in vivo. Therefore, it is suggested that this method should support the elucidation of the in vivo structure and function of the knee joint and might be applied to clinical practice.     

Investigation of laminar appearance of articular cartilage by means of magnetic resonance microscopy

Magnetic resonance (MR) images and relaxation and diffusion maps of articular cartilage were obtained to explain discrepancies in its MR appearance. Porcine specimens were studied only by MR microscopy. For human specimens a combination of MR microscopy and large-scale MR imaging was used. Common features in the laminar structures of human and porcine samples are described. It was found that the decay of transverse magnetization was nonexponential with a rapidly decaying component which prevented construction of reliable proton-density maps. Dependence of T₂ values on the orientation of specimens in the magnetic field as well as magnetization transfer experiments supported the previous suggestions about a significant role of dipolar interaction with protons of collagen in the laminar appearance of articular cartilage. The loss of the laminar structure induced by rotation of the human cartilage specimen around the axis normal to its surface demonstrated nonuniform angular distribution of the collagen fibers within the layer.     

The zonal architecture of human articular cartilage described by T2 relaxation time in the presence of Gd-DTPA2-

The depth-wise variation of T(2) relaxation time is known to reflect the collagen network architecture in cartilage, while the delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) technique is sensitive to tissue proteoglycan (PG) concentration. As the cartilage PG content varies along the tissue depth, the depth-dependent accumulation of the contrast agent may affect the inherent T(2) of cartilage in a nonconstant manner. Therefore, T(2) and dGEMRIC are typically measured in separate MRI sessions. In the present in vitro MRI study at 9.4 T, depth-wise T(2) profiles and collagenous zone thicknesses as determined from T(2) maps in the absence and presence of Gd-DTPA(2-) (T(2) and T(2Gd), respectively) were compared in samples of intact human articular cartilage (n=65). These T(2) measures were further correlated with birefringence (BF) of polarized light microscopy (PLM) to quantify the ability of MRI to predict the properties of the collagen fibril network. The reproducibility of the T(2) measurement in the current setup was also studied. Typical tri-laminar collagen network architecture was observed both with and without Gd-DTPA(2-). The inverse of BF (1/BF) correlated significantly with both T(2) and T(2Gd) (r=0.91, slope=0.56 and r=0.90, slope=0.63), respectively. The statistically significant linear correlations between zone thicknesses as determined from T(2) and T(2Gd) were r=0.55 (slope=0.49), r=0.74 (slope=0.71) and r=0.95 (slope=0.94) for superficial, middle and deep tissue zones, respectively. Reproducibility of the T(2) measurement was worst for superficial cartilage. Consistent with PLM, T(2) and T(2Gd) measurements reveal highly similar depth-dependent information on collagen network in intact human cartilage. Thus, dGEMRIC and T(2) measurements in one MRI session are feasible for intact articular cartilage in vitro.     

关节软骨内胶原纤维各向异性的红外光谱学显微成像研究

福尔马林溶液对于固化关节软骨组织、防止在长时间测量过程中组织的分解退化起到很好的作用,但对福尔马林溶液浸泡后软骨组织的结构变化(固化)过程及其胶原纤维各向异性的改变却鲜有研究。采用傅里叶变换红外光谱显微成像技术与偏振技术相结合的方法,通过关节软骨内胶原纤维(蛋白)的红外光谱特征吸收峰(Amide Ⅰ,Amide Ⅱ带)的吸光度随福尔马林溶液浸泡时间及偏光角度的变化来研究福尔马林溶液对软骨组织结构即胶原纤维各向异性的影响,并利用与各向异性方程拟合得到的决定系数(R2)对胶原蛋白纤维各向异性程度进行表征。研究发现,关节软骨Amide Ⅰ和Amide Ⅱ带的各向异性随着福尔马林浸泡时间的增长而愈加明显(Amide Ⅰ带变化尤为明显),说明福尔马林溶液中甲醛分子诱发了胶原蛋白分子新的交联,最终获得较好的固化效果,有利于关节软骨的各向异性分析。本研究将为今后关节软骨样本的制备、储存及各向异性研究提供参考。     

Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging

We investigated the role of collagen in the magnetization transfer (MT) effect in contrast to other macromolecules. By means of phantoms made of collagen, chondroitin sulfate (CS) and albumin, MR parameters have been optimized in order to reduce the acquisition time and improve the sensitivity, as well as to minimize the contributions from CS and albumin to the MT induced signal attenuation. The same method was used to study cartilage ex vivo (bovine articular and nasal cartilage plugs) and in vivo (goat knee femoral chondyle). In phantom samples, the MT signal attenuation depended on the collagen concentration while contributions from the other macromolecules were found to be minimal. In average, analysis of MT images revealed a 25%, 35% and 30% signal attenuation in 10% w/v type I collagen gels, cartilage plugs, and cartilage from the weight-bearing areas of the goat knee, respectively. Biochemical data revealed that treatment of cartilage plugs with bacterial collagenase led to collagen depletion and correspondingly to a decrease of the MT response. In contrast, trypsin-induced proteoglycan loss in cartilage plugs did not alter the MT effect. A significant correlation was observed between the collagen content in these plugs and their respective MT ratios and the rate constant k for the exchange process bound versus free water. Finally, data obtained from in vivo MT measurement of the goat knee demonstrated that intra-articular injection of papain might not only cause degradation of proteoglycans but also a change in collagen integrity in a dose-dependent manner. We conclude that in vivo measurement of MT ratios gives quantitative and qualitative information on the collagen status and may be applied for the routine evaluation of normal and abnormal articular cartilage.     

The structure of the fibrous tissue on the articular surface of the temporal bone in the monkey (Macaca mulatta)

The articulating surface of bones which ossify in mesenchyme, like the mandible, is covered by a layer of dense, fibrous tissue. The purpose of the present study was to examine the structure of the fibrous tissue on the surface of the articular surface of the temporal bone in the monkey. Young Rhesus monkeys (Macaca mulatta) were perfused with glutaraldehyde-paraformaldehyde. The specimens were demineralized in 0.5M EDTA. Small pieces of fibrous tissue and underlying bone were dissected out and processed for light and electron microscopy. The mandibular fossa is shallower and the articular eminence flatter in the monkeys as compared to humans. The articular part of the temporal bone is covered by a layer of avascular, soft tissue extending from the surface to the underlying bone. The tissue can be divided into three zones which gradually merge into one another. The zone facing the articular cavity consists of dense, fibrous tissue with layers of collagen fibers, oriented parallel to the articular surface, but at angles to each other. Fibers thought to be elastic fibers oriented parallel with the collagen fibers are also observed, particularly close to the surface, and their function is probably to impart resilience to the fibrous articular tissue. Between the fibers scattered cells with an ample rough endoplasmic reticulum are present. A thin layer of granular appearance is often observed on the surface. This layer may be of importance in joint lubrication. The second zone is more cell rich and the cells have long slender cellular processes and are surrounded by a dense collagenous matrix with an irregular orientation. These cells are probably precursor for the underlying cartilage but, not for the cells in the outer articular layer. In the third zone next to the bone the fibrous tissue gradually turns into cartilage. The cartilagenous zone is narrow, sometimes absent and is replaced by bone tissue. In some areas chondroclasts are observed, with forming osteons with osteoid seams. These observations indicate that remodeling is taking place and that cartilage is replaced by bone. The three zones observed correspond to findings in the mandibular condyle, but the zones are not as constant and distinct as in the condyle, and this reflects the adaptive role of the temporal bone in the growth of the temporomandibular joint.     

Evaluation of water content by spatially resolved transverse relaxation times of human articular cartilage

Non-invasive assessment of cartilage properties, specifically water content, could prove helpful in the diagnosis of early degenerative joint diseases. Transverse relaxation times T(2) of human articular cartilage (34 cartilage slices of three donors) were measured on a pixel-by-pixel basis in a clinical whole body MR system in vitro. In vivo feasibility to measure quantitative T(2) maps was shown for human patellar cartilage. The relaxation times of cartilage with collagen in the radial zone oriented perpendicular to the magnetic field increased from approximately 10 ms near the bone to approximately 60 ms near the articular surface. Cartilage water content of the tibial plateau and femoral condyles could be determined from the correlation with T(2) (R(2) = 0.71) with an error of approximately 2 wt.%. In vivo, directional variation would need to be considered. If confirmed in vivo, T(2) measurements could potentially serve as a non-invasive tool for the evaluation of the status and distribution of water content in articular cartilage.     

Low-field one-dimensional and direction-dependent relaxation imaging of bovine articular cartilage

The structure of articular cartilage is separated into three layers of differently oriented collagen fibers, which is accompanied by a gradient of increasing glycosaminoglycan (GAG) and decreasing water concentration from the top layer towards the bone interface. The combined effect of these structural variations results in a change of the longitudinal and transverse relaxation times as a function of the distance from the cartilage surface. In this paper, this dependence is investigated at a magnetic field strength of 0.27 T with a one-dimensional depth resolution of 50 μm on bovine hip and stifle joint articular cartilage. By employing this method, advantage is taken of the increasing contrast of the longitudinal relaxation rate found at lower magnetic field strengths. Furthermore, evidence for an orientational dependence of relaxation times with respect to an axis normal to the surface plane is given, an observation that has recently been reported using high-field MRI and that was explained by preferential orientations of collagen bundles in each of the three cartilage zones. In order to quantify the extent of a further contrast mechanism and to estimate spatially dependent glycosaminoglycan concentrations, the data are supplemented by proton relaxation times that were acquired in bovine articular cartilage that was soaked in a 0.8 mM aqueous Gd++ solution.     

Differences in Cartilage Repair between Loading and Unloading Environments in the Rat Knee

We investigated the histopathological and immunohistochemical effects of loading on cartilage repair in rat full-thickness articular cartilage defects. A total of 40 male 9-week-old Wistar rats were studied. Full-thickness articular cartilage defects were created over the capsule at the loading portion in the medial condyle of the femur. Twenty rats were randomly allocated into each of the 2 groups: a loading group and a unloading group. Twenty rats from these 2 groups were later randomly allocated to each of the 2 groups for evaluation at 1 and 2 weeks after surgery. At the end of each period, knee joints were examined histopathologically and immunohistochemically. In both groups at 1 and 2 weeks, the defects were filled with a mixture of granulation tissue and some remnants of hyaline cartilage. The repair tissue was not stained with toluidine blue in both groups. Strong staining of type I collagen was observed in the repair tissue of both groups. The area stained with type I collagen was smaller in the unloading group than in the loading groups, and the stained area was smaller at 2 weeks than at 1 week. In the staining for type II collagen, apparent staining of type II collagen was observed in the repair tissue of both groups at 1 week. At 2 weeks, there was a tendency toward a higher degree of apparent staining in the loading group than in the unloading group. Accordingly, these results indicated that loading and unloading in the early phase of cartilage repair have both merits and demerits.     

Transverse relaxation mechanisms in articular cartilage

Relaxation rates in the rotating frame (R1rho) and spin-spin relaxation rates (R2) were measured in articular cartilage at various orientations of cartilage layer to the static magnetic field (B0), at various spin locking field strengths and at two different static magnetic field strengths. It was found that R1rho in the deep radial zone depended on the orientation of specimens in the magnet and decreased with increasing the spin locking field strength. In contrast, R1rho values in the transitional zone were nearly independent of the specimen orientation and the spin locking field strength. Measurements of the same specimens at 2.95 and 7.05 T showed an increase of R1rho and most R2 values with increasing B0. The inverse B0 dependence of some R2 values was probably due to a multicomponent character of the transverse magnetization decay. The experiments revealed that the dominant T1rho and T2 relaxation mechanism at B0 < or = 3 T is a dipolar interaction due to slow anisotropic motion of water molecules in the collagen matrix. On average, the contribution of scalar relaxation due to rapid proton exchange in femoral head cartilage at 2.95 T is about 6% or less of the total R1rho at the spin locking field of 1000 Hz.     

Raman analysis of proteoglycans simultaneously in bone and cartilage

Bone and cartilage are connective tissues with distinct organic matrix (collagen and non‐collagenous proteins) composition facilitating their biological function. Proteoglycans (PGs), a member of the non‐collagenous proteins fulfill functions that are determined by both their core protein and their glycosaminoglycan chains. The purpose of the present study was to identify Raman bands that are representative of PG concentration and may be used in both bone and cartilage tissues. To achieve this goal, we analyzed a series of reference PGs and collagens, as well as turkey leg tendon to verify the laser polarization independency of the identified bands. Additionally, the applicability of these bands in both cartilage and bone tissue simultaneously was tested in a healthy femoral head by Raman imaging and hierarchical cluster analysis to describe the distribution of PGs at the micron level from articular cartilage to subchondral bone. The results of the study show that the Raman band ~1375 cm⁻¹ can be used as a PGs marker band in both cartilage and bone. Moreover, articular cartilage has a lower content of organic matrix (mostly type II collagen), while the middle and deep transitional zone haves a higher concentration of PGs. The calcified cartilage is characterized by a lower content of PGs and total organic matrix (estimated from the integrated area of the amide III band). Copyright © 2014 John Wiley & Sons, Ltd.     

Vibrational spectroscopy of articular cartilage

Articular cartilage is a connective tissue that is located at the ends of long bones. Type II collagen, proteoglycans, water, and chondrocytes are the main constituents of articular cartilage. Osteoarthritis, the most common joint disease in the world, causes degenerative changes in articular cartilage tissue. Fourier transform infrared, Raman, and near infrared spectroscopic techniques offer versatile tools to assess biochemical composition and quality of articular cartilage. These vibrational spectroscopic techniques can be used to broaden our understanding about the compositional changes during osteoarthritis, and they also hold promise in disease diagnostics. In this article, the current literature of articular cartilage spectroscopic studies is reviewed.     

Optimization and validation of a rapid high-resolution T1-w 3D FLASH water excitation MRI sequence for the quantitative assessment of articular cartilage volume and thickness

In view of follow up, survey and development of therapeutic strategies for osteoarthritis where cartilage deterioration plays an important role, a non invasive, reliable and quantitative assessment of the articular cartilage is desirable. The currently available high resolution T(1)-weighted (T1-w) 3D FLASH pulse sequences with frequency selective fat suppression are very time consuming. We have 1) optimized a high resolution T1-w 3D FLASH water excitation (WE) sequence for short acquisition time and cartilage visualization, and 2) validated this sequence for cartilage volume and thickness quantification. The spectral fat presaturation was replaced by selective water excitation. The flip angle of the WE sequence was optimized for the contrast to noise (C/N(cart)) ratio of cartilage. Sagittal datasets (voxel size: 0.31 x 0.31 x 2 mm(3)) of the knees of nine healthy volunteers were acquired both, with the 3D FLASH WE (17.2/6.6/30 degrees ) sequence (WE) and a previously validated 3D FLASH fat saturated (42/11/30 degrees ) sequence (FS). For validation of the WE sequence, cartilage volume, mean and maximal cartilage thickness of the two sequences were compared. Reproducibility was assessed by calculating the coefficient of variation (COV %) of 4 consecutive WE data sets in the volunteers. The acquisition time was reduced from 16'30" (FS) down to 7'14" for the WE sequence. Image contrast and visualization of the cartilage was very similar, but delineation of the basal layer of the cartilage was slightly improved with the WE sequence. A flip angle of 30 degrees provided the best C/N(cart) ratios (WE). Reproducibility (COV) was between 1.9 and 5.9%. Cartilage volume and thickness agreed within 4% between FS and WE sequence. The WE sequence allows for rapid, valid and reproducible quantification of articular cartilage volume and thickness, prerequisites for follow-up examinations. The reduced acquisition time (50% of FS) enables routine clinical application and thus may contribute to a broader assessment of osteoarthritis.     

<Emphasis Type="Italic">T</Emphasis><Subscript>1</Subscript>–<Emphasis Type="Italic">T</Emphasis><Subscript>2</Subscript> Correlation and Biopolymer Diffusion Within Human Osteoarthritic Cartilage Measured with Nuclear Magnetic Resonance

Cartilage is a load-bearing tissue that provides smooth articulation during motion of human joints like the knee and hip. Cartilage deterioration in the form of osteoarthritis (OA) causes painful joint motion in more than 100 million patients worldwide, and thus there is great interest in improving our understanding of cartilage to further clinical treatment. Previous studies have examined many aspects of cartilage mechanics, including the flow of interstitial water and repulsion of neighboring glycosaminoglycan chains. However, the contributions of specific molecules to overall tissue properties remain unclear. In this study, we use nuclear magnetic resonance (NMR) diffusometry and relaxometry to examine the molecular dynamics of water and cartilage polymers in OA human articular cartilage. To our knowledge, this is the first identification of two macromolecular populations corresponding to collagen and proteoglycan in human cartilage through their diffusive properties. Further, we performed NMR T ₁–T ₂ correlation studies on human cartilage and observed two populations of water distinguished by differing NMR relaxation corresponding to a solid-like component and a liquid-like component. These results provide fundamental insight on the water behavior and polymeric interactions that drive the functional mechanics of cartilage. This study provides a basis to both expand our understanding of basic cartilage mechanics and provide molecular dynamics data for design of novel biomaterials to improve joint health.     

Dynamics of the biopolymers in articular cartilage studied by magic angle spinning NMR

To understand the viscoelastic properties of cartilage tissue and for the development of tissue-engineered cartilage, we have studied the physicochemical properties of bovine nasal and pig articular cartilage by¹³C nuclear magnetic resonance (NMR) methods. The major macromolecular components of cartilage can be investigated individually by applying¹³C high-resolution (HR) NMR with scalar decoupling (for the polysaccharide component) and solid-state NMR with dipolar decoupling (for the collagen component). Partially resolved NMR spectra of the cartilage polysaccharides can be obtained by HR¹³C NMR indicating that these polysaccharides are highly mobile. Resonance lines have been assigned to chondroitin sulfate, the most mobile component of cartilage. To characterize time scales of molecular motions, we have measuredT ₁ andT ₂ relaxation times as a function of temperature and analyzed these data by means of a broad distribution of molecular correlation times. Typical correlation times for the large amplitude motions of chondroitin sulfate are of the order of 0.1–10 ns. For the detection and dynamical characterization of the cartilage collagen cross-polarization magic angle spinning (CP MAS) and high-power decoupling are indispensable.¹³C CP MAS spectra of cartilage are dominated by resonances from rigid collagen, while only low-intensity signals from the polysaccharides are observed. The good sensitivity at a magnetic field strength of 17.6 T allows the site-specific investigation of cartilage collagen dynamics by two-dimensional NMR methods. The cartilage collagen is essentially rigid with low-amplitude segmental motions on the fast time scale. Considering the high water content of cartilage and the almost isotropic mobility of the chondroitin sulfate molecules it is remarkable how little this affects the collagen dynamics. The dynamics of cartilage macromolecules is broadly distributed from almost completely rigid to highly mobile, which lends cartilage its mechanical strength and shock-absorbing properties.     

Diameter distributions of collagenous tissues in relation to sex. A quantitative ultrastructural study

A number of factors have been implicated in the regulation of collagen fibril diameter. Electron microscopy analysis was used to investigate the role of sex on fibril diameter. Female mouse skin collagen fibrils' mean diameter values were significantly smaller than those from the male, independent of age. In addition, the female rabbit collagen fibrils had a marked decrease in the mean diameter compared to male, in all the tissues examined (skin, liver, and bone). These data suggest that the collagen fibril diameter is related to sex.     

Effects of the Thermal Environment on Articular Chondrocyte Metabolism: A Fundamental Study to Facilitate Establishment of an Effective Thermotherapy for Osteoarthritis

Aim: To facilitate establishment of an effective thermotherapy for osteoarthritis (OA), we investigated the effects of the thermal environment on articular chondrocyte metabolism in vitro. Methods: Chondrocytes were isolated from porcine knee joints, and cultured at 32°C, 37°C and 41°C. Cell proliferation and viability were assessed at Days 2, 4 and 8. In addition, TdT-mediated dUTP nick end labeling (TUNEL) assay was performed at Day 3 to determine the proportion of apoptotic chondrocytes. Analysis of genes specific for factors related to the cartilage extracellular matrix (ECM), cartilage destruction, and cartilage protection was performed at Day 2. Furthermore, evaluation of heat stress tolerance, and heat shock protein 70 (HSP70) mRNA expression and protein synthesis was performed at Day 2 and 3, respectively. Results: Cell proliferation was more at 37°C than at 32°C and 41°C. Cell viability and the number of TUNEL-positive cells were not affected until Day 8 and 3, respectively. The expression of the ECM-related genes was up-regulated at higher temperature. The expression of MMP13, a type II collagen destructive enzyme, and that of TIMP1 and TIMP2, which are MMP inhibitors, were up-regulated at higher temperatures. Finally, the chondrocytes cultured at 41°C may acquire heat stress tolerance, in part, due to the up-regulation of HSP70, and may inhibit apoptosis induced by various stresses, which is observed in OA. Conclusions: The thermal environment affects articular chondrocyte metabolism, and a heat stimulus of approximately 41°C could enhance chondrocyte anabolism and induce heat stress tolerance.     

Assessment of interstitial water content of articular cartilage with T1 relaxation

The interstitial water content typically increases in the early degeneration of articular cartilage. Previously, T₂ relaxation has been related to water content, yet it is known to be strongly affected by the collagen orientation. Articular cartilage plugs from the bovine patella, femur and tibia (N=20) were mapped for T₁ and T₂ at 9.4 T to test the ability of T₁ relaxation to reflect cartilage water content. As a reference, water and proteoglycan (PG) contents were determined. Significant (P<.01) linear associations were demonstrated between the relaxation rates and tissue water content (R₁: r=−.81, R₂: r=−.60) and PG content (R₁: r=.75). After adjustment for the tissue water content, partial correlation analysis did not show significant associations between the relaxation rates and tissue PG content. After the effect of PGs was removed, significant (P<.05) linear correlation between the relaxation rates and tissue water content (R₁: r=−.48, R₂: r=−.50) was observed. Thus, the spin-lattice relaxation rate is proposed to provide a biomarker for water content in articular cartilage.     

A study of the relationship between molecular biomarkers of joint degeneration and the magnetic resonance-measured characteristics of cartilage in 16 symptomatic knees

We used quantitative magnetic resonance (MR) imaging to determine if relationships exist between proposed molecular biomarkers for degenerative joint disease (DJD) and structural characteristics of articular cartilage. Subjects were eight male and eight female volunteers diagnosed with osteoarthritis. Magnetic resonance images of the symptomatic knee were taken and blood samples were drawn. Concentrations of serum cartilage oligomeric matrix protein (COMP) and cleaved collagen neoepitope were compared to cartilage volume and cartilage T2, respectively, in four compartments of the tibiofemoral joint. A significant, negative correlation was found between serum COMP and medial tibia volume in the male subject group (rho=-.738, P=.037). A significant, positive correlation (rho=.881, P=.0039) was found between serum COMP and lateral femur volume in the female subject group. In both groups, positive correlations were found between serum C2C and cartilage T2, which were significant in two compartments of the male group (rho=.714, P=.047; rho=.738, P=.037) and similarly strong, but not statistically significant (rho=.750, P=.052), in one compartment of the female group. We identify strong and biologically relevant correlations between two proposed molecular biomarkers for DJD and MR measures of symptomatic knees of a small number of arthritic patients. Our findings support the hypothesis that cartilage molecular biomarkers reflect the molecular processes of cartilage degeneration and loss.     

Dependencies of multi-component T2 and T1ρ relaxation on the anisotropy of collagen fibrils in bovine nasal cartilage

Both NMR spectroscopy and MRI were used to investigate the dependencies of multi-component T2 and T1ρ relaxation on the anisotropy of bovine nasal cartilage (BNC). The non-negative least square (NNLS) method and the multi-exponential fitting method were used to analyze all experimental data. When the collagen fibrils in nasal cartilage were oriented at the magic angle (55°) to the magnetic field B0, both T2 and T1ρ were single component, regardless of the spin-lock field strength or the echo spacing time in the pulse sequences. When the collagen fibrils in nasal cartilage were oriented at 0° to B0, both T2 and T1ρ at a spin-lock field of 500 Hz had two components. When the spin-lock field was increased to 1000 Hz or higher, T1ρ relaxation in nasal cartilage became a single component, even when the specimen orientation was 0°. These results demonstrate that the specimen orientation must be considered for any multi-component analysis, even for nasal cartilage that is commonly considered homogenously structured. Since the rapidly and slowly relaxing components can be attributed to different portions of the water population in tissue, the ability to resolve different relaxation components could be used to quantitatively examine individual molecular components in connective tissues.